The field of proteomics has evolved significantly in the last decade, with several technologies now available to assay protein biomarkers in clinical studies. In this article, we compare three popular approaches for proteomics – mass spectrometry, ELISA, and the Olink® Proximity Extension Assay (PEA) – and highlight their strengths and limitations.
Mass Spectrometry
Assays based on mass spectrometry can cast a wide net for detecting proteins and reveal information about their structure, post-translational modifications, and binding partners. Mass spectrometry involves several labor-intensive steps: proteins are first digested into peptides, which are then separated on a column, ionized into a vapor state, fragmented, and detected by mass spectrometers. The technique also requires a high amount of starting material, and therefore, is best suited to quantify highly abundant proteins in a complex sample.
Advantages
- High specificity
- Identifies protein sequences and structures, including post-translational modifications
- Compatible with many sample types
Limitations
- Requires a large amount of sample input
- Low throughput
- Time-intensive and expensive
ELISA
Another widely used method for protein detection is ELISA, which stands for enzyme-linked immunosorbent assay. This technique is typically performed in 96-well polystyrene plates and involves immobilization and detection of antigens of interest using complementary antibodies. A few variations of ELISA have been developed, such as direct ELISA, indirect ELISA, and sandwich ELISA. The most popular method for protein quantification is sandwich ELISA, which receives its name due to the antigen being bound between two specific antibodies. It’s highly sensitive but has low throughput since one protein is assayed at a time.
Advantages
- High sensitivity and specificity
- Medium-to-high throughput for sample processing
Limitations
- Requires a large amount of sample input
- Detects only one protein at a time
Olink Proximity Extension Assay
The limitations of traditional approaches have led to the evolution of new technologies for protein biomarker discovery that are highly sensitive and high throughput. One prominent example is Olink’s PEA. Each protein is detected by a matched pair of antibodies that are conjugated to partially complementary oligonucleotides (see figure below). When both antibodies bind to the target, an amplicon can be generated, which is subsequently measured by quantitative real-time PCR (qPCR) or next generation sequencing (NGS). The use of dual antibody recognition and DNA annealing eliminates cross-reactive events since only matched DNA reporter pairs can hybridize to produce an amplicon. This enables very high-throughput, multiplexed immunoassays without loss of sensitivity or specificity. It also requires minimal volumes of biological material.
Advantages
- High sensitivity and specificity
- Identifies up to 384 proteins at the same time
- High throughput at reasonable costs
- Low sample input required
Limitations
- Currently validated with serum and plasma samples, although other sample types can be used
Comparison of Assays
Below we highlight key similarities and differences between mass spectrometry, ELISA, and Olink PEA to help you decide which is the best approach for your experiments.
| Mass Spectrometry | ELISA | Olink PEA | |
|---|---|---|---|
Technology | Mass-to-charge analysis of peptide fragments | Antibody-based | Antibodies coupled to oligonucleotides |
| Throughput | Low: one sample at a time; possible scale-up at extra cost | Medium: up to 96 samples per plate | Medium: up to 88 samples per plate |
| Multiplexing | High: depends on protein abundance | Low: one protein at a time | Medium: up to 88 samples per plate |
| Sensitivity | Low | High | High |
| Quantification | Absolute and relative | Absolute and relative | Absolute and relative |
| Sample Input | ~150 µL (highly concentrated) | ~100 µL | ~1 µL |
| Price | Cost-effective for a few samples | Cost-effective for 96 samples | Cost-effective for ≥88 samples |
Conclusion
The tools for protein biomarker detection have increased in recent years, giving preclinical and clinical researchers greater options. All approaches have tradeoffs, but the Olink platform provides a balanced combination of multiplexing, specificity, sensitivity, throughput, and sample volume. Read our tech note for a deeper dive into Olink PEA and to learn how this technology can support clinical trials.